Side-chain cleavage of cholesterol by cytochrome 450 CYP11A1 (i.e., classical pathway of steroidogenesis) is not implicated in the biosynthesis of MBG, as it is for endogenous ouabain. Previous studies in toads, known to produce bufadienolides, demonstrated that administration of labeled cholesterol and cholanic acid, but not of progesterone, the product of cholesterol side-chain cleavage, resulted in the incorporation of a radioactive label into bufadienolide molecules. Because the extra-hepatic synthesis of bile acids has recently been described, we hypothesized that in placenta, in which high levels of MBG are detected in preeclampsia, bufadienolides could be synthesized from bile acids. In human trophoblast JEG-3 cells, we examined the impact of post-transcriptional silencing of two genes, encoding sterol 27-hydrolase (CYP27A1), an initiating enzyme for synthesis of cholanic acid from cholesterol, and CYP11A1, an enzyme which controls side-cleavage of cholesterol into pregnenolone, on production of MBG and progesterone. Levels of steroids were measured in cell culture media. In JEG-3 cells, silencing of CYP11A1 resulted in 90% decrease of the protein amount (Western blot), in 80% decrease of mRNA (qPCR), and reduced production of progesterone by 77%, but did not affect production of MBG as compared to non-transfected and mock-transfected cells. Silencing of CYP27A1, accompanied by 75% reduction in CYP27A1 protein, and by 65% reduction in mRNA level, conversely, did not affect production of progesterone, but suppressed production of MBG by 80% vs. that in non-transfected and mock-transfected cells. Thus, in human trophoblast cells, bufadienolide cardiotonic steroid MBG is synthesized from cholesterol via bile acid pathway which represents a novel pathway of hormone biosynthesis.